Several mechanisms have been established by which E. coli can cause diarrhea. Some strains are enterotoxigenic and induce fluid secretion by colonizing the small bowel and by producing heat-labile or heat-stable enterotoxins. Other strains are enteroinvasive and mimic shigellae by invading and damaging colonic epithelial cells. A third group of strains, designated enteropathogenic E. coli (EPEC), are incriminated as agents of epidemic and endemic diarrheal disease in infants but have not been classified as enterotoxigenic or enteroinvasive. Recent studies from this laboratory revealed that EPEC and other E. coli enteric isolates do make a cell-associated toxin that is indistinguishable from the toxin of Shigella dysenteriae 1 (Shiga toxin). Two lines of evidence suggest that this E. coli Shiga-like toxin could be a virulence determinant in EPEC strains. First, the purified Shiga-like toxin of E. coli is a potent cytotoxin (1 pg = 1 HeLa cell cytotoxic dose) and is also an enterotoxin that can elicit fluid secretion in a rabbit ligated ileal segment. Second, the destruction of gut epithelial cell microvilli that is observed on small bowel biopsy of human infants with EPEC diarrhea is confined to regions of dense E. coli adherence, suggesting that an E. coli cell-associated cytotoxin might mediate the damage. The long range goals of this project are to analyze molecular mechanisms of toxinogenesis in E. coli and to determine if E. coli Shiga-like toxin has an essential role in the pathogenesis of EPEC diarrhea. Our studies will characterize the relationships between structure, antigenicity, and function of Shiga-like toxin and analyze the genetic basis for control of toxinogenesis at a molecular level. The results of these experiments will be applied for development of isogenic strains of enteropathogenic E. coli which differ only with respect to Shiga-like toxin production, and these strains will be compared for virulence in experimental animals. Our studies will contribute to an improved understanding of the molecular biology of Shiga-like toxin and its role in pathogenesis in gastrointestinal infections by E. coli. Such information is essential for assessing the potential contribution of antitoxic immunity in developing a successful method for immunizing against EPEC diarrhea.